Pharmacodynamic characterization of gemcitabine cytotoxicity in an in vitro cell culture bioreactor system.

PURPOSE: Gemcitabine, a pyrimidine nucleoside, is approved for the treatment of non-small cell lung cancer, pancreatic carcinoma, and breast cancer. Chemotherapy regimens are determined experimentally with static tissue culture systems, animal models, and in Phase I clinical trials. The aim of this study was to assess for gemcitabine-induced cell death following infusion of drug under clinically-relevant conditions of infusion rate and drug exposure in an in vitro bioreactor system. METHODS: To estimate an appropriate harvest time for cells from the bioreactor after drug treatment, we estimated the temporal relationship between gemcitabine treatment for 1 h and cell death at a later time point with monolayer growth assays (i.e., static culture). Afterward, 5.3 mg gemcitabine was infused over 0.5 h in the bioreactor, followed by mono-exponential decay, simulating patient concentration-time profiles (n = 4). Controls were run with drug-free media (n = 4). Cells were harvested from the bioreactor at a later time point and assessed for cell death by flow cytometry. RESULTS: According to monolayer growth assay results, cytotoxicity became more apparent with increasing time. The E Max for cells 48 h after treatment was 50% and after 144 h, 93% (P = 0.022; t test), while flow cytometry showed complete DNA degradation by 120 h. Gemcitabine was infused in the bioreactor. The gemcitabine area under the concentration-time curve (AUC) was 56.4 microM h and the maximum concentration was 87.5 +/- 2.65 microM. Flow cytometry results were as follows: the G1 fraction decreased from 65.1 +/- 4.91 to 28.6 +/- 12% (P = 0.005) and subG1 increased from 14.1 +/- 5.28 to 42.6 +/- 9.78% (P = 0.004) relative to control. An increase in apoptotic cells was observed by TUNEL assay. CONCLUSIONS: The in vitro bioreactor system will be expanded to test additional cell lines, and will serve as a useful model system for assessing the role of drug pharmacokinetics in delivery of optimized anticancer treatment.

Short versus continuous gemcitabine treatment of non-small cell lung cancer in an in vitro cell culture bioreactor system.

Five-year survival for non-small cell lung cancer is 15%. Gemcitabine is a nucleoside analogue that inhibits ribonucleotide reductase and interferes with DNA replication. In this study, we sought to compare short versus continuous infusion gemcitabine in an in vitro bioreactor system using pharmacokinetic-guided dosing. Gemcitabine was infused over either 0.5 or 2.5h to produce concentration-time profiles that mimic those measured in biological samples (i.e., patient plasma). The effects of gemcitabine on the growth and survival of H2009 cells were examined using trypan blue staining, cell cycle analysis, TUNEL assay, and clonogenic assay. Data were analyzed with two ways analysis of variance. Maximum gemcitabine (Cmax) concentrations during the short infusion were 51.2+/-10.4 microM and for the continuous, 14.8+/-2.93 microM. Steady-state concentrations during the continuous infusions were 14.9+/-2.90 microM. Gemcitabine treatment resulted in a decrease for G1 fraction relative to controls. G2/M, subG1 and TUNEL were higher following gemcitabine relative to controls. Survival was approximately 20-fold higher following the short infusion compared with the continuous infusion (p = 0.0085). In conclusion, gemcitabine infused by this novel method induced apoptosis after both the short and continuous infusions, and long-term survival was significantly diminished following continuous compared with the short infusion.

Enhancement of tumor thermal therapy using gold nanoparticle-assisted tumor necrosis factor-alpha delivery.

Tumor necrosis factor-alpha (TNF-alpha) is a potent cytokine with anticancer efficacy that can significantly enhance hyperthermic injury. However, TNF-alpha is systemically toxic, thereby creating a need for its selective tumor delivery. We used a newly developed nanoparticle delivery system consisting of 33-nm polyethylene glycol-coated colloidal gold nanoparticles (PT-cAu-TNF-alpha) with incorporated TNF-alpha payload (several hundred TNF-alpha molecules per nanoparticle) to maximize tumor damage and minimize systemic exposure to TNF-alpha. SCK mammary carcinomas grown in A/J mice were treated with 125 or 250 microg/kg PT-cAu-TNF-alpha alone or followed by local heating at 42.5 degrees C using a water bath for 60 minutes, 4 hours after nanoparticle injection. Increases in tumor growth delay were observed for both PT-cAu-TNF-alpha alone and heat alone, although the most dramatic effect was found in the combination treatment. Tumor blood flow was significantly suppressed 4 hours after an i.v. injection of free TNF-alpha or PT-cAu-TNF-alpha. Tumor perfusion, imaged by contrast enhanced ultrasonography, on days 1 and 5 after treatment revealed perfusion defects after the injection of PT-cAu-TNF-alpha alone and, in many regions, complete flow inhibition in tumors treated with combination treatment. The combination treatment of SCK tumors in vivo reduced the in vivo/in vitro tumor cell survival to 0.05% immediately following heating and to 0.005% at 18 hours after heating, suggesting vascular damage-mediated tumor cell killing. Thermally induced tumor growth delay was enhanced by pretreatment with TNF-alpha-coated gold nanoparticles when given i.v. at the proper dosage and timing.

Synergistic effects of radiation and beta-lapachone in DU-145 human prostate cancer cells in vitro.

It has been reported that beta-lapachone (beta-lap), a bioreductive anti-cancer drug, synergistically interacts with ionizing radiation and that the sensitivity of cells to beta-lap is closely related to the activity of NAD(P)H:quinone oxidoreductase 1 (NQO1). Here we report the results of our studies of mechanisms underlying the synergistic interaction of beta-lap and radiation in killing cancer cells using the DU-145 human prostate cancer cell line. The clonogenic cell death caused by the combination of radiation and beta-lap was synergistic when beta-lap was administered 0-10 h after irradiation but not when it was given before irradiation. The expression and activity of NQO1 increased significantly and remained elevated for longer than 12 h after 4 Gy irradiation, suggesting that the long-lasting elevation of NQO1 sensitized the cells to beta-lap. Studies with split-dose irradiation demonstrated that beta-lap given immediately after irradiation effectively inhibited sublethal radiation damage (SLD) repair. Taken together, these results lead us to conclude that the synergistic interaction between beta-lap and radiation in killing cells is the result of two distinct mechanisms: First, radiation sensitizes cells to beta-lap by up-regulating NQO1, and second, beta-lap sensitizes cells to radiation by inhibiting SLD repair. The combination of beta-lap and radiotherapy is potentially promising modality for the treatment of cancer in humans.

Simultaneous inhibition of the receptor kinase activity of vascular endothelial, fibroblast, and platelet-derived growth factors suppresses tumor growth and enhances tumor radiation response.

We have investigated the effect of simultaneous inhibition of multiple angiogenic growth factor signaling pathways on tumor growth, tumor blood perfusion, and radiation-induced tumor-growth delay using SU6668, an inhibitor of the receptor-tyrosine kinase activity of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF). The SCK mammary carcinoma, FSaII fibrosarcoma, and CFPAC human pancreatic carcinoma were grown s.c. in the hind leg of A/J mice, C3H mice, and Balb/cAnNCrl-nuBr nude mice, respectively. Daily i.p. injection of 100 mg/kg of SU6668 markedly suppressed the growth of these three tumor types. SU6668 also markedly prolonged the survival time of host mice bearing SCK tumors, which appeared to be caused by a reduction of metastatic tumor growth in the lung. There was little or no change in normal tissue blood perfusion, whereas in SCK tumors the perfusion decreased by 50% at 1 h after a single i.p. injection of SU6668, slightly recovered at 4 h, and completely recovered by 8 h. Interestingly, the tumor blood flow was significantly increased above the baseline level 24 h after SU6668 injection. After extended daily i.p. injections of SU6668, the tumor blood flow in all of the three tumor types studied was markedly decreased compared with control. The observed effects of this drug on tumor blood perfusion may partially explain the effectiveness of the compound in suppressing tumor growth and extending survival of tumor-bearing mice. We also observed that daily SU6668 administration and a single dose of 15 Gy of X-irradiation was significantly more effective than either treatment alone in suppressing tumor growth. Our results suggest that SU6668 increased the radiosensitivity of tumor blood vessels. We conclude that SU6668 is a potent therapeutic agent potentially useful to suppress tumor growth and enhance the response of tumors to radiotherapy.

Preferential action of arsenic trioxide in solid-tumor microenvironment enhances radiation therapy.

PURPOSE: To investigate the effect of arsenic trioxide, Trisenox (TNX), on primary cultures of endothelial cells and tumor tissue under varying pH and pO(2) environments and the effects of combined TNX and radiation therapy on experimental tumors. METHODS AND MATERIALS: Human dermal microvascular endothelial cells were cultured in vitro and exposed to TNX under various combinations of aerobic, hypoxic, neutral, or acidic conditions, and levels of activated JNK MAP kinase were assessed by Western blotting. FSaII fibrosarcoma cells grown in the hind limb of female C3H mice were used to study the effect of TNX on tumor blood perfusion and oxygenation. The tumor-growth delay after a single or fractionated irradiation with or without TNX treatment was assessed. RESULTS: A single intraperitoneal injection of 8 mg/kg TNX reduced the blood perfusion in FSaII tumors by 53% at 2 hours after injection. To increase the oxygenation of the tumor vasculature during TNX treatment, some animals were allowed to breathe carbogen (95% O(2)/5% CO(2)). Carbogen breathing alone for 2 hours reduced tumor perfusion by 33%. When carbogen breathing was begun immediately after TNX injection, no further reduction occurred in tumor blood perfusion at 2 hours after injection. In vitro, TNX exposure increased activity JNK MAP kinase preferentially in endothelial cells cultured in an acidic or hypoxic environment. In vivo, the median oxygenation in FSaII tumors measured at 3 or 5 days after TNX injection was found to be significantly elevated compared with control tumors. Subsequently, radiation-induced tumor-growth delay was synergistically increased when radiation and TNX injection were fractionated at 3-day or 5-day intervals. CONCLUSIONS: Trisenox has novel vascular-damaging properties, preferentially against endothelium in regions of low pH or pO(2), which leads to tumor cell death and enhancement of the response of tumors to radiotherapy.

Assessing pH and oxygenation in cryotherapy-induced cytotoxicity and tissue response to freezing.

The microenvironmental pH and oxygenation is known to influence tumor cell response to heat, radiation, photodynamic and even chemotherapy. We have studied the previously untested influence of acidity and hypoxia on tumor and endothelial cell sensitivity to freezing. In addition, we have measured changes in oxygenation in vivo in murine FSaII fibrosarcomas after freeze injury. A low pH or low oxygenation environment was found to increase the sensitivity of tumor and endothelial cells to freezing at -20 degrees C or -40 degrees C in vitro. However, low pH and low oxygenation combined did not further increase cryosensitivity of the cells. In vivo, tumor oxygenation after freeze injury was studied immediately or 1-3 days after a standard freezing protocol was applied to FSaII tumors ranging from 250-500 mm3 grown in the rear-limb of C3H mice. Tumor oxygenation at the edge of the iceball was found to transiently increase 1-2 hours after freezing. At 1-3 days after freezing, a treatment that delayed FSaII tumor growth by approximately 1.5-fold, the mean tumor oxygenation was significantly increased by up to 2.5-fold from a control level of 5 mmHg partial pressure of oxygen (pO2), especially at the periphery of the tumor. We conclude that manipulation of pH or oxygenation has potential to increase the anti-tumor effects of minimally invasive cryosurgical techniques. Furthermore, the dynamic changes in oxygenation after freeze injury in vivo suggests value in combining cryotherapy with treatments dependent on oxygenation levels. Ultimately, these may be routes to more reliable treatment response with fewer recurrences.

Study of arsenic trioxide-induced vascular shutdown and enhancement with radiation in solid tumor.

PURPOSE: Arsenic trioxide (ATO) has been reported to be an effective chemotherapeutic agent for acute promyelocytic leukemia (APL), and, recently, anti-tumor effect has been demonstrated in solid tumors. However, little is known about the mechanism of action of the ATO effect on solid tumor. We investigated the anti-vascular effect of ATO and the potential of combining ATO with radiation therapy. MATERIALS AND METHODS: We studied the anti-vascular effect of ATO and radiosensitization of squamous cell carcinoma (SCC) VII murine tumors of C3H mice. The anti-vascular effect was examined using magnetic resonance imaging(MRI), and radiosensitivity was studied by clonogenic assay and tumor growth delay. Histopathological changes of the tumors after various treatments were also observed with hematoxylin and eosin (H&E) staining. RESULTS: Necrosis and blood flow changes in the central region of tumors in the hind limbs of the animals were observed on T2-weighted imaging after an i.p. injection of 8 mg/kg of ATO alone. ATO exposure followed by radiation decreased the clonogenic survival of SCC VII cells compared with either treatment alone. Tumor growth delay after 10-20 Gy of radiation alone was increased slightly compared with control tumors, but the combination of ATO injection 2 hours before exposure to 20 Gy of radiation significantly prolonged tumor growth delay by almost 20 days. CONCLUSION: The results suggest that ATO and radiation can enhance the radiosensitivity of solid tumor.

Pharmacodynamic modeling of sequence-dependent antitumor activity of insulin-like growth factor blockade and gemcitabine.

Agents that block insulin-like growth factor (IGF) signaling are under investigation in clinical trials. Antitumor effects are likely to be enhanced when combined with other agents, but administration sequence effects on activity are not well-described. Three breast cancer cell lines (MCF-7, MDA-MB-231, and Hs-578T) were treated with Gemcitabine and small molecule receptor tyrosine kinase inhibitor cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]1-(2-phenyl-quinolin-7-yl)-imidazo [1,5-a]pyrazin-8-ylamine (PQIP) as single agents and then in combination in the forward (Gemcitabine followed by PQIP) and reverse (PQIP followed by Gemcitabine) sequences. Antitumor effects were assessed longitudinally by Bayesian analysis using WinBUGS. The pharmacodynamic model adequately predicted the observed data. The differences in the cell-kill rate constants for the forward vs. reverse sequence ranged from 0.11 to 0.64 (day(-1)), and statistical significance was generally dependent on cell line and PQIP concentration. These data indicate that treatment with Gemcitabine first, followed by PQIP is superior to the reverse sequence in vitro.

Vascular disrupting agent arsenic trioxide enhances thermoradiotherapy of solid tumors.

Our previous studies demonstrated arsenic trioxide- (ATO-) induced selective tumor vascular disruption and augmentation of thermal or radiotherapy effect against solid tumors. These results suggested that a trimodality approach of radiation, ATO, and local hyperthermia may have potent therapeutic efficacy against solid tumors. Here, we report the antitumor effect of hypofractionated radiation followed by ATO administration and local 42.5 °C hyperthermia and the effects of cisplatin and thermoradiotherapy. We found that the therapeutic efficacy of ATO-based thermoradiotherapy was equal or greater than that of cisplatin-based thermoradiotherapy, and marked evidence of in vivo apoptosis and tumor necrosis were observed in ATO-treated tumors. We conclude that ATO-based thermoradiotherapy is a powerful means to control tumor growth by using vascular disruption to augment the effects of thermal and radiation therapy.

Combinatorial pharmacologic effects of gemcitabine and its metabolite dFdU.

Recent evidence has shown that the gemcitabine metabolite, dFdU, is pharmacologically active. Though less potent, dFdU has a longer half-life and could potentiate or antagonize the activity of gemcitabine. Hence, studies were undertaken to evaluate the combined effects. Following chemical synthesis, an improved purification procedure for dFdU was developed (80 % yield; >99 % purity). Zebrafish phenotype-based embryo screens revealed no acute toxicity after gemcitabine or dFdU treatment. Only gemcitabine affected zebrafish development in a dose-dependent manner. Synergy or antagonism for the combination was not observed. Antitumor effects for dFdU were dose dependent. Antagonism was tumor cell-line dependent and did not depend on formation of the intracellular active metabolite of gemcitabine, suggesting that the drug-metabolite interaction occurs later. These studies highlight a platform for testing the pharmacologic activity for anticancer agent and metabolite combinations. Such analyses are expected to provide insight into the beneficial or harmful effect(s) of metabolites towards parent drug activity.

Cap-dependent translation blockade and fixed dose-rate gemcitabine: interaction in an in vitro bioreactor system.

Translation initiation commences with the binding of eIF-4F to the mRNA 5'-end cap. eIF-4F binds the cap structure via its eIF-4E subunit, which is the rate-limiting step for the initiation of translation. This pathway can be inhibited by 4E-binding proteins (4E-BPs). The present study investigated prolonged gemcitabine infusion in combination with reduced eIF-4E function on NSCLC cell viability in an in vitro bioreactor system. To assess attachment to the hollow fibers, cells with dominant active 4E-BP1 were first analyzed by scanning electron microscopy. Cells were treated with 0.5- or 2.5h (fixed dose rate) infusion (same total dose), simulating human plasma gemcitabine concentration-time profiles. An interaction was observed between fixed dose rate infusion gemcitabine and presence of dominant active 4E-BP1. We conclude that cap-dependent translation blockade and fixed dose rate infusion gemcitabine treatment results in a significant interaction affecting cell viability in vitro.

Exposure-response relationships for oxaliplatin-treated colon cancer cells.

Data are lacking for an optimal infusion length for oxaliplatin administered intraperitoneally. Our objectives were to establish the roles of hyperthermia and an effective length of oxaliplatin treatment in maximizing antitumor activity. SW620 cells were treated for 0.5 vs. 2 h and at 37 vs. 42 degrees C. Cytotoxicity, cell cycle analysis, subG1 and survival were assessed with the MTT assay, flow cytometry and the clonogenic assay. The IC50 for cells treated at 37 degrees C was 2.90+/-0.83 microg/ml and at 42 degrees C, 1.99+/-0.66 microg/ml (P=0.14). The Emax for 37 degrees C was 93.9+/-2.57% and for 42 degrees C, 97.8+/-1.59% (P=0.05). The subG1 fraction did not differ between cells treated at 37 and 42 degrees C (P=0.12). The IC50 for the cells treated for 0.5 h was 10.6+/-0.60 microg/ml and for 2 h, 2.80+/-1.70 microg/ml (P=0.02). The Emax for 0.5 h was 87.9+/-5.13% and for 2 h, 96.6+/-3.35% (P=0.09). SubG1 for 0.5 h was 8.24+/-1.33% and for 2 h, 15.8+/-2.45% (P=0.02). Clonogenic assays demonstrated diminished survival when treated with low concentrations (10 microg/ml) of oxaliplatin combined with heat treatment (P=0.017) for 2 h, but not 0.5 h. Similar clonogenic assay experiments were performed with the oxaliplatin-resistant WiDr cell line, and differences in survival following oxaliplatin and heat treatment were again observed for 2 h, but not for 0.5 h (P=0.002). Drug treatment for 2 h of both SW620 and WiDr cell lines is superior to treatment for 0.5 h. Cell kill effects are reliant on treatment length; hence, the choice of time exposure must be made with a view to maintaining a balance between the cell kill effects and the clinical feasibility of treating the patient.

Hyperthermic enhancement of tumor radiosensitization strategies.

Local hyperthermia of living tissue can cause significant increases in blood flow and oxygenation depending on time-temperature history. Increases in perfusion of the abnormal and insufficient vasculature found in solid tumors may increase tumor oxygenation, thereby increasing the radiation sensitivity of the tumor. We hypothesized that local heating of tumor would increase the oxygenation of the tumor tissue and allow other oxygenating agents to further modify tumor oxygenation and radiation response. In the present study the effect of moderate temperature hyperthermia (MTH) at 41.5-42.5 degrees C for 30-60 min, 250 mg/kg nicotinamide, or carbogen breathing (95% O2/5% CO2) on the radiation sensitivity of FSaII murine fibrosarcomas or R3230 AC rat adenocarcinomas was studied. Individually, these treatments increased the tumor cell sensitivity to single dose 10-15 Gy X-irradiation by 1-5 fold on average, as measured by the in vivo/in vitro tumor excision assay. The combination of tumor MTH with nicotinamide or carbogen breathing increased the radiation sensitivity by 3-5 fold in FSaII tumors and 10-30 fold in R3230 tumors with varying levels of statistical significance. Finally, the triple combination of adjuvant MTH, nicotinamide and carbogen breathing increased the radiation-induced cell death in FSaII tumors to a similar extent as the dual combinations of MTH, nicotinamide or heat, carbogen breathing. However, in R3230 AC tumors the triple adjuvant combination significantly increased radiation-induced cell killing compared to all other dual adjuvant treatments (p < 0.04). To interrogate the mechanism by which heating alters tumor physiology, nitric oxide production in tumor and endothelial cells in culture and tumor tissue after heating was studied. Heating caused an increase in nitric oxide production over a 24 h period after treatment. Subsequently, inhibiting the enzymatic production of NO with L-NAME was found to increase heat-induced growth delay of FSaII tumors. The cause and effect of increased nitric oxide production and the response of the tumor vasculature to heat are discussed in the context of the tumor radiosensitization achieved by heating, carbogen breathing and nicotinamide.

Anginex synergizes with radiation therapy to inhibit tumor growth by radiosensitizing endothelial cells.

We have demonstrated that the designed peptide anginex displays potent antiangiogenic activity. The aim of our study was to investigate the effect of anginex on established tumor vasculature as an adjuvant to radiation therapy of solid tumors. In the MA148 human ovarian carcinoma athymic mouse model, anginex (10 mg/kg) in combination with a suboptimal dose of radiation (5 Gy once weekly for 4 weeks) caused tumors to regress to an impalpable state. In the more aggressive SCK murine mammary carcinoma model, combination of anginex and a single radiation dose of 25 Gy synergistically increased the delay in tumor growth compared to the tumor growth delay caused by either treatment alone. Immunohistochemical analysis also demonstrated significantly enhanced effects of combined treatment on tumor microvessel density and tumor or endothelial cell proliferation and viability. In assessing physiologic effects of anginex, we observed a reduction in tumor perfusion and tumor oxygenation in SCK tumors after 5-7 daily treatments with anginex with no reduction in blood pressure. To test anginex as a radiosensitizer, additional studies using SCK tumors were performed. Three daily i.p. injections of anginex were able to enhance the effect of 2 radiation doses of 10 Gy, resulting in 50% complete responses, whereas the known antiangiogenic agent angiostatin did not enhance the radiation response of SCK tumors. Mechanistically, it appears that anginex functions as an endothelial cell-specific radiosensitizer because anginex showed no effect on in vitro radiosensitivity of SCK or MA148 tumor cells, whereas anginex significantly enhanced the in vitro radiosensitivity of 2 endothelial cell types. This work supports the idea that the combination of the antiangiogenic agent anginex and radiation may lead to improved clinical outcome in treating cancer patients.